Coinfection with Enterohepatic Helicobacter species can ameliorate or promote Helicobacter pylori-induced gastric pathology in C57BL/6 mice

To investigate how different enterohepatic Helicobacter species (EHS) influence Helicobacter pylori gastric pathology, C57BL/6 mice were infected with Helicobacter hepaticus or Helicobacter muridarum, followed by H. pylori infection 2 weeks later. Compared to H. pylori-infected mice, mice infected with H. muridarum and H. pylori (HmHp mice) developed significantly lower histopathologic activity index (HAI) scores (P < 0.0001) at 6 and 11 months postinoculation (MPI). However, mice infected with H. hepaticus and H. pylori (HhHp mice) developed more severe gastric pathology at 6 MPI (P = 0.01), with a HAI at 11 MPI (P = 0.8) similar to that of H. pylori-infected mice. H. muridarum-mediated attenuation of gastritis in coinfected mice was associated with significant downregulation of proinflammatory Th1 (interlukin-1beta [Il-1β], gamma interferon [Ifn-γ], and tumor necrosis factor-alpha [Tnf-α]) cytokines at both time points and Th17 (Il-17A) cytokine mRNA levels at 6 MPI in murine stomachs compared to those of H. pylori-infected mice (P < 0.01). Coinfection with H. hepaticus also suppressed H. pylori-induced elevation of gastric Th1 cytokines Ifn-γ and Tnf-α (P < 0.0001) but increased Th17 cytokine mRNA levels (P = 0.028) at 6 MPI. Furthermore, mRNA levels of Il-17A were positively correlated with the severity of helicobacter-induced gastric pathology (HhHp>H. pylori>HmHp) (at 6 MPI, r2 = 0.92, P < 0.0001; at 11 MPI, r2 = 0.82, P < 0.002). Despite disparate effects on gastritis, colonization levels of gastric H. pylori were increased in HhHp mice (at 6 MPI) and HmHp mice (at both time points) compared to those in mono-H. pylori-infected mice. These data suggest that despite consistent downregulation of Th1 responses, EHS coinfection either attenuated or promoted the severity of H. pylori-induced gastric pathology in C57BL/6 mice. This modulation was related to the variable effects of EHS on gastric interleukin 17 (IL-17) responses to H. pylori infection.     

17β-estradiol and tamoxifen prevent gastric cancer by modulating leukocyte recruitment and oncogenic pathways in Helicobacter pylori-infected INS-GAS male mice

Helicobacter pylori infection promotes male predominant gastric adenocarcinoma in humans. Estrogens reduce gastric cancer risk and previous studies showed that prophylactic 17β-estradiol (E2) in INS-GAS mice decreases H. pylori-induced carcinogenesis. We examined the effect of E2 and tamoxifen (TAM) on H. pylori-induced gastric cancer in male and female INS-GAS mice. After confirming robust gastric pathology at 16 weeks postinfection (WPI), mice were implanted with E2, TAM, both E2 and TAM, or placebo pellets for 12 weeks. At 28 WPI, gastric histopathology, gene expression, and immune cell infiltration were evaluated and serum inflammatory cytokines measured. After treatment, no gastric cancer was observed in H. pylori-infected males receiving E2 and/or TAM, whereas 40% of infected untreated males developed gastric cancer. E2, TAM, and their combination significantly reduced gastric precancerous lesions in infected males compared with infected untreated males (P < 0.001, 0.01, and 0.01, respectively). However, TAM did not alter female pathology regardless of infection status. Differentially expressed genes from males treated with E2 or TAM (n = 363 and n = 144, Q < 0.05) associated highly with cancer and cellular movement, indicating overlapping pathways in the reduction of gastric lesions. E2 or TAM deregulated genes associated with metastasis (PLAUR and MMP10) and Wnt inhibition (FZD6 and SFRP2). Compared with controls, E2 decreased gastric mRNA (Q < 0.05) and serum levels (P < 0.05) of CXCL1, a neutrophil chemokine, leading to decreased neutrophil infiltration (P < 0.01). Prevention of H. pylori-induced gastric cancer by E2 and TAM may be mediated by estrogen signaling and is associated with decreased CXCL1, decreased neutrophil counts, and downregulation of oncogenic pathways.     

Helicobacter hepaticus--induced liver tumor promotion is associated with increased serum bile acid and a persistent microbial-induced immune response

Chronic microbial infection influences cancer progression, but the mechanisms that link them remain unclear. Constitutive androstane receptor (CAR) is a nuclear receptor that regulates enzymes involved in endobiotic and xenobiotic metabolism. CAR activation is a mechanism of xenobiotic tumor promotion; however, the effects of chronic microbial infection on tumor promotion have not been studied in the context of CAR function. Here, we report that CAR limits the effects of chronic infection-associated progression of liver cancer. CAR knockout (KO) and wild-type (WT) male mice were treated with or without the tumor initiator diethylnitrosamine (DEN) at 5 weeks of age and then orally inoculated with Helicobacter hepaticus (Hh) or sterile media at 8 weeks of age. At approximately 50 weeks postinoculation, mice were euthanized for histopathologic, microbiological, molecular, and metabolomic analyses. Hh infection induced comparable hepatitis in WT and KO mice with or without DEN that correlated with significant upregulation of Tnfα and toll receptor Tlr2. Notably, DEN-treated Hh-infected KO mice exhibited increased numbers of liver lobes with dysplasia and neoplasia and increased multiplicity of neoplasia, relative to similarly treated WT mice. Enhanced tumor promotion was associated with decreased hepatic expression of P450 enzymes Cyp2b10 and Cyp3a11, increased expression of Camp, and increased serum concentrations of chenodeoxycholic acid. Together, our findings suggest that liver tumor promotion is enhanced by an impaired metabolic detoxification of endobiotics and a persistent microbial-induced immune response.     

Gastrin Is an Essential Cofactor for Helicobacter-Associated Gastric Corpus Carcinogenesis in C57BL/6 Mice

We have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in H. felis-infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice.Gastric cancer remains the second leading cause of cancer-related mortality in the world, although its incidence and mortality rates have been decreasing in the United States over the past 70 years.^1,2,3 The risk of developing gastric adenocarcinoma is strongly associated with Helicobacter pylori infection, which is gradually disappearing from western societies. Despite the overall decline in gastric cancer prevalence, the treatment of stomach cancer remains a challenging clinical problem, since most patients who undergo surgical resection develop regional or distant recurrences and the overall 5-year survival rate for gastric cancer patients remains around 20% in western countries.³H. pylori, first identified in the gastric antrum of patients with active chronic gastritis and peptic ulcers,⁴ is now recognized as the major cause of gastric cancer, and has been classified as a group I carcinogen by World Health Organization.^5,6 H. pylori infection causes persistent chronic gastritis, which in susceptible individuals may progress to atrophy, intestinal metaplasia, dysplasia, and finally, intestinal-type gastric cancer. This sequence, commonly referred to as Correa’s cascade, is considered the primary histological pathway for the development of intestinal type of gastric cancer,⁷ and is both initiated and promoted by H. pylori infection.It has generally been recognized that H. pylori infection results in a mild (1.5- to 2-fold elevation) hypergastrinemia that occurs early on in the course of the infection in many individuals. Given the known properties of gastrin as a mucosal growth factor, hypergastrinemia was postulated to be a factor promoting the development of gastric cancer. Indeed, previous studies have suggested a possible association between hypergastrinemia, Helicobacter infection, and gastric cancer.^8,9,10,11,12 Therefore, to study the role of gastrin and the potential mechanisms involved in gastric carcinogenesis, we developed a mouse model of gastric cancer through the generation of insulin-gastrin (INS-GAS) transgenic mice that overexpressed human amidated gastrin. In the absence of Helicobacter infection, INS-GAS mice on an FVB/N genetic background exhibited mild hypergastrinemia in association with elevated gastric acid secretion and an increased parietal cell number at 1 to 3 months of age. With increasing age, the INS-GAS mice showed progressive loss of parietal cells and significant changes in the corpus, including hypochlorhydria, gastric atrophy, metaplasia, and dysplasia. At 20 months of age, INS-GAS mice developed invasive gastric cancer.⁹ The gastric cancer phenotype was accelerated by gastric Helicobacter spp. infection, and lesion severity was more profound in male INS-GAS mice.¹⁰ The cause of this gender-specific incidence was due in part to ovarian-dependent estrogen production, since H. pylori infected ovariectomized female INS-GAS mice also developed severe gastric neoplasia, and 17beta-estradiol treatment significantly suppressed this phenotype.¹²However, determining the role of gastrin in predisposing individuals to gastric cancer has not been straightforward. Some H. pylori-infected patients have lower levels of gastrin and acid secretion relative to non-infected healthy persons, and hypochlorhydria probably plays an important role in the carcinogenic process through altered bacterial colonization along with changes in nitrite levels.¹³ Gastrin-deficient mice on a mixed C57BL6/129Sv background developed spontaneous gastric antral tumors when maintained under conventional housing conditions at 12 months of age, while C57BL/6 wild-type and somatostatin-deficient mice did not develop tumors.¹⁴ The authors concluded that neoplastic transformation of the antrum does not require gastrin, and that gastrin may actually suppress the development of gastric antral tumors. In addition, there have been other genetic models reported, such as the gp130^757F/F mouse, which do not appear to be dependent on gastrin for tumor development.¹⁵ It has been difficult to reconcile these observations regarding the influence of gastrin on gastric cancer, given the different genetic backgrounds, housing conditions, and Helicobacter infection status. Thus, the purpose of this study is to examine the effect of gastrin in Helicobacter-associated gastric carcinogenesis using hypergastrinemic (INS-GAS) mice and gastrin deficient (GAS-KO) mice on a uniform C57BL/6 background and housed under SPF conditions.     

Concurrent Helicobacter bilis Infection in C57BL/6 Mice Attenuates Proinflammatory H. pylori-Induced Gastric Pathology

Because coinfections can alter helicobacter gastritis, we investigated whether enterohepatic Helicobacter bilis modulates Helicobacter pylori gastritis in C57BL/6 mice. Thirty mice per group were sham dosed, H. bilis or H. pylori infected, or H. bilis infected followed in 2 weeks by H. pylori and then evaluated at 6 and 11 months postinfection (mpi) for gastritis and premalignant lesions. Compared to H. pylori-infected mice, H. bilis/H. pylori-infected mice at 6 and 11 mpi had less severe gastritis, atrophy, mucous metaplasia and hyperplasia (P < 0.01) and, additionally, at 11 mpi, less severe intestinal metaplasia and dysplasia (P < 0.05). H. bilis/H. pylori-infected mice at 11 mpi exhibited less Ki67 labeling of proliferating epithelial cells, reduced numbers of FoxP3⁺ T-regulatory (T_REG) cells, and lower FoxP3⁺ mRNA levels than did H. pylori-infected mice (P < 0.05). Proinflammatory interleukin-1β (IL-1β), gamma interferon, and tumor necrosis factor alpha mRNA levels were attenuated in H. bilis/H. pylori-infected mice at 6 and 11 mpi (P < 0.01), although anti-inflammatory IL-10, IL-13, and transforming growth factor β1 mRNA levels were not consistently impacted by H. bilis coinfection. Decreased pathology in H. bilis/H. pylori-infected mice correlated with higher gastric H. pylori colonization at 6 mpi (P < 0.001) and lower Th1-associated immunoglobulin G2c responses to H. pylori at 6 and 10 mpi (P < 0.05). We hypothesized that reduced pathology in H. bilis/H. pylori-infected mice was due to H. bilis-primed T_REG cells in the lower bowel that migrated to the gastric compartment and inhibited Th1 responses to subsequent H. pylori infection. Thus, H. pylori-induced gastric lesions may vary in mouse models of unknown enteric helicobacter infection status and, importantly, variable sequelae to human H. pylori infection, particularly in developing countries, may occur where coinfection with lower bowel helicobacters and H. pylori may be common.Helicobacter pylori, first isolated by Warren and Marshall, induces a persistent infection and gastritis and is known to colonize the stomach of over 50% of the human population (2). In a subset of infected individuals, H. pylori is linked to the development of peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. It has been classified by the World Health Organization as a class I carcinogen (25). It is not clear why some individuals infected with H. pylori develop serious disease, while others do not. Host and environmental factors, as well as the virulence properties of H. pylori, appear to play an important role in determining disease outcome (17, 52, 60). Poor socioeconomic conditions promote early acquisition and infection with H. pylori and infection rates often approach >90% in these populations. Interestingly, some African countries with especially high prevalence rates of infection have lower-than-expected rates of gastric cancer. This paradox has been referred to as “the African Enigma” (24). The low incidence of gastric cancer has been linked to endemic parasites, diet, poor cancer registry data, and the low pathogenicity of some H. pylori strains (3a, 12, 31a).Like humans, mice respond immunologically to infectious agents with a repertoire of memory T cells that respond most efficiently after antigen priming (28) but also appear to modulate host responses to unrelated infections and likely are also effective in disease caused by organisms sharing common antigens. These cross-reactive T cells, when activated, not only modulate the immune response but also determine the eventual outcome of heterologous infections. This host immune response is often referred to as heterologous immunity (47). This phenomenon has been studied to a limited extent in mouse models of gastric helicobacter pathogenesis that have had varied pathological outcomes. In a C57BL/6 mouse model of H. felis gastritis, coinfection with an enteric helminth, Heligmosomoides polygyrus, stimulated a Th2 response that attenuated Th1-promoted gastric pathology (11). In contrast, in BALB/c mice which have a Th2-biased response to gastric helicobacter infection resulting in no discernible gastritis, coinfection with Toxoplasma gondii promoted a robust Th1 immune response, resulting in a progressive helicobacter-associated gastritis, gastric atrophy, and metaplasia (50). Recently, we have demonstrated that the colitis induced by Citrobacter rodentium resulted in a prolonged recovery of the disease in C57BL/6 mice when the animals were coinfected with H. hepaticus (34). It is also known that host immune responses resulting from infections with atypical mycobacteria can influence how mice or humans respond immunologically to BCG vaccination (9, 62). These examples of heterologous immunity suggest that disease outcomes can be impacted by modulation of Th1 and Th2 inflammatory responses (37).Subclinical lower bowel helicobacter infections are prevalent worldwide in mouse colonies; however, the persistent infection in certain inbred strains of mice often elicits demonstrable pathology (53). In susceptible mouse strains, enterohepatic helicobacters cause inflammatory bowel disease, colonic adenocarcinoma, hepatitis, cholecystitis, and hepatocellular carcinoma (8, 31, 33, 57). Non-H. pylori helicobacters are increasingly cited in association with human diarrheal disease, particularly in developing countries, as well as with hepatobiliary diseases in humans (10, 12, 13, 22, 38). These observations of enterohepatic helicobacter-associated disease in humans and the common occurrence of enteric helicobacter infections in mice suggest that helicobacter coinfections could impact murine studies involving H. pylori pathogenesis, vaccine strategies, and antimicrobial modalities. Thus, we initiated an experiment to ascertain whether coinfection with H. bilis, an enterohepatic helicobacter with a wide host range (10), could impact the progression of H. pylori-induced gastric disease and inflammatory responses in C57BL/6 mice (10, 12, 19, 46).     

DNA repair is indispensable for survival after acute inflammation

More than 15% of cancer deaths worldwide are associated with underlying infections or inflammatory conditions, therefore understanding how inflammation contributes to cancer etiology is important for both cancer prevention and treatment. Inflamed tissues are known to harbor elevated etheno-base (ε-base) DNA lesions induced by the lipid peroxidation that is stimulated by reactive oxygen and nitrogen species (RONS) released from activated neutrophils and macrophages. Inflammation contributes to carcinogenesis in part via RONS-induced cytotoxic and mutagenic DNA lesions, including ε-base lesions. The mouse alkyl adenine DNA glycosylase (AAG, also known as MPG) recognizes such base lesions, thus protecting against inflammation-associated colon cancer. Two other DNA repair enzymes are known to repair ε-base lesions, namely ALKBH2 and ALKBH3; thus, we sought to determine whether these DNA dioxygenase enzymes could protect against chronic inflammation-mediated colon carcinogenesis. Using established chemically induced colitis and colon cancer models in mice, we show here that ALKBH2 and ALKBH3 provide cancer protection similar to that of the DNA glycosylase AAG. Moreover, Alkbh2 and Alkbh3 each display apparent epistasis with Aag. Surprisingly, deficiency in all 3 DNA repair enzymes confers a massively synergistic phenotype, such that animals lacking all 3 DNA repair enzymes cannot survive even a single bout of chemically induced colitis.